Table I.
NADPH oxidation and ebastine hydroxylation rates of CYP2J2 constructs in detergent solubilized and membrane bound systems. Each of the four constructs were incorporated into Nanodiscs, reconstituted with lipids or solubilized in 0.1% CHAPS and used to measure NADPH consumption and hydroxyebastine product formation. The coupling efficiency was calculated by taking the ratio of ebastine hydroxylation measured using LC/MS/MS versus the NADPH Oxidation monitored via UV-Vis spectroscopy at 340 nm
| CYP2J2 Constructs | Substrate-Free NADPH Rate(nmol/min/nmol) | Ebastine NADPH Rate (nmol/min/nmol) | Ebastine Hydroxylation Rate (nmol/min/nmol) | Coupling Efficiency |
|---|---|---|---|---|
| CYP2J2-CPR Nanodiscs | ||||
| M2SMG | 9.2 ± 1.5 | 20.3 ± 0.9 | 3.8 ± 0.7 | 17.0 ± 0.8 |
| M2D5HG | 7.8 ± 0.8 | 21.3 ± 0.4 | 3.7 ± 0.6 | 17.1 ± 0.7 |
| M2D19HG | 12.9 ± 0.7 | 27.1 ± 1.6 | 4.5 ± 0.4 | 16.5 ± 0.9 |
| M2D34G | 12.2 ± 0.3 | 21.2 ± 0.3 | 2.9 ± 0.1 | 13.6 ± 1.3 |
| CYP2J2-POPC Reconstituted | ||||
| M2SMG | 34.0 ± 1.7 | 69 ± 2.0 | 4.3 ± 0.2 | 6.1 ± 0.2 |
| M2D5HG | 31.8 ± 2.8 | 72 ± 6.4 | 4.4 ± 0.3 | 6.1 ± 0.7 |
| M2D19HG | 38.9 ± 3.3 | 88 ± 6.3 | 5.4 ± 0.1 | 6.2 ± 0.4 |
| M2D34G | 65.1 ± 1.3 | 121 ± 8.5 | 3.1 ± 0.7 | 2.5 ± 0.3 |
| CYP2J2-Detegrent | ||||
| M2SMG | 21.5 ± 1.7 | 50.6 ± 3.7 | 1.6 ± 0.2 | 3.2 ± 0.1 |
| M2D5HG | 23.2 ± 1.0 | 44.9 ± 6.1 | 1.4 ± 0.3 | 3.1 ± 0.2 |
| M2D19HG | 27.3 ± 1.5 | 72.2 ± 7.3 | 2.6 ± 0.9 | 3.5 ± 0.9 |
| M2D34G | 28.0 ± 1.4 | 142.5 ± 18.6 | 2.4 ± 0.5 | 1.7 ± 0.1 |